Electrophoresis is one of the most widely used techniques in biotechnology laboratories for analyzing mixtures of macromolecules, whether they be proteins, nucleic acids, carbohydrates or other species. Many electrophoretic procedures use a gel as a separation medium, and in many cases, greater sensitivity, range and versatility can be achieved by the use of a gradient gel, in which a characteristic of the gel is varied with the distance along one or more axes of the gel. Porosity gradients, for example, are useful in separating mixtures of a large number of components whose molecular weights vary over a wide range, since high molecular weight species require a relatively high gel porosity for separation from each other while low molecular weight species require a relatively low porosity. In gels containing a denaturant, a gradient is often formed in the concentration of the denaturant, particularly when the gel is used for separations of nucleic acids for DNA sequencing.
Gradients are useful in capillary or tube-shaped gels as well as slab gels. In slab gels, the gradients may be either parallel or perpendicular to the direction of migration, with useful results obtained in both cases. For example, denaturant gradients which are perpendicular to the direction of electrophoretic migration are useful in certain separations of nucleic acids to distinguish mutant genes from wild-type genes. Denaturant gradients which are parallel to the direction of separation are often used as the second stage of a two-dimensional separation, following a first stage where separation is performed in a constant gel. Linear gradients are most often used although nonlinear gradients are preferred in some cases. Stepwise variations are also useful.
The gradient may be formed by distributing the gel monomers and other components used in forming the gel between two source mixtures differing in concentration, then drawing from the two source mixtures at proportions which vary with time. For porosity gradients, the two source mixtures will vary in the concentration of the gel monomer, the crosslinking agent if one is used, or both. For denaturant gradients, the source mixtures will vary in the concentration of the denaturant. The method is simple in principle, but difficult to apply in a manner which results in a high degree of precision and reproducibility. In particular, for the laboratory technician who produces gradient gels for immediate use in the laboratory itself, there is a need for a simple device which can be used in the laboratory and which has a minimum of moving parts, yet is reliably precise and reproducible from one gel to the next.
These and other problems associated with prior art gradient formers are addressed by the present invention.